Chimeric CrylE Delta Endotoxin and Methods of Controlling Insects

ABSTRACT

The present invention relates to a chimeric δ endotoxin protein Cry 1E of SEQ ID No. 1 with extraordinarily high insecticidal property and a chimera gene of SEQ ID No. 2 encoding the said chimeric protein, and also a method of treating insect infested plants using said chimera protein.

CLAIM OF PRIORITY

This application is a continuation (and claims the benefit of priorityunder 35U.S.C. §120) of U.S. application Ser. No. 11/062,225, filed Feb.18, 2005, which application is a divisional (and claims the benefit ofpriority under 35 U.S.C. §120) of U.S. application Ser. No. 10/107,581,filed Mar. 27, 2002. The disclosure of the prior applications areconsidered part of (and are incorporated by reference in) the disclosureof this application.

FIELD OF THE PRESENT INVENTION

The present invention relates to a chimeric δ endotoxin protein Cry 1Eof SEQ ID No. 1 with extraordinarily high insecticidal property and achimera gene of SEQ ID No. 2 encoding the said chimeric protein, andalso a method of treating insect infested plants using said chimeraprotein.

BACKGROUND OF THE INVENTION

Damage due to insects costs billions of dollars annually in form of croplosses and in the expense of keeping these pests under control. Thelosses caused by pests in agricultural production environments includedecrease in crop yield, poor crop quality, increased harvesting costs,and loss to health and environment.

Reference may be made to Hofte H. and Whiteley H. R., 1989,“Insecticidal crystal protein of Bacillus thuringiensis”, Microbiol.Rev. 53: 242-255, wherein Bacillus thuringiensis (B.t.) is a ubiquitousgram-positive spore-forming soil bacterium, known for its ability toproduce parasporal crystalline inclusions during sporulation. Theseinclusions consist of proteins known as crystal proteins or Cry proteinsor δ-endotoxins, which exhibit insecticidal activity, particularlyagainst larvae of insect species in orders lepidoptera, diptera andcoleoptera. Proteins with toxicity to insects of orders hymenoptera,homoptera, orthoptera, mallophaga; nematodes; mites and protozoans havealso been mentioned in literature (Feitelson J. S., 1993, “The Bacillusthuringiensis family tree”, 63-71, In L. kim ed. Advanced engineeredpesticides. Marcel Dekker. Inc., New York., N.Y. and Feitelson et al.,1992, “Bacillus thuringiensis: insects and beyond”, Bio/Tech. 10:271-275; may be sited for this). Several strains of Bacillusthuringiensis (B.t.) have been identified with different host spectraand classified into different subspecies or serotypes on the basis offlagellar antigens. Pasteur Institute, France has catalogued 55different flagellar serotypes and 8 non-flagellated biotypes. Thereference may be made to Schnepf et al., 1998, “Bacillus thuringiensisand its pesticidal crystal proteins”, Microbiol. Mol. Biol. Riv. 62:775-806, wherein several B.t. toxin-coding genes have been cloned,sequenced, characterised and recombinant DNA-based products to have beenproduced and approved for commercial use. Through the employment ofgenetic engineering techniques, new approaches have been developed fordelivering these B.t. toxins to agricultural environments, including theuse of the genetically engineered crops and the stabilised intactmicrobial cells as δ-endotoxin delivery vehicles (Gaertner, F. H., KimL., 1988, TIBTECH 6: 54-57). Thus, δ-endotoxin genes coding for proteinstargeted to kill hosts, especially pests and insects that cause economiclosses are becoming commercially valuable.

Commercial use of B.t. pesticides in a given crop environment is limitedbecause a given δ-endotoxin shows toxicity to a narrow range of targetpests. Preparations of the spores and crystals of B. thuringiensissubsp. kurstaki have been used for many years as commercial insecticidesagainst lepidopteran pests. For example, B. thuringiensis var. kurstakiHD-1 produces several δ-endotoxins, and is therefore toxic to arelatively broader range of lepidopteran insects. However, formulationsbased on the known δ-endotoxins, including B.t.k. HD-1 are not effectiveagainst some of the important crop pests, like Spodoptera sp. that alsobelong to order lepidoptera. Other species of B.t., namely israelensisand tenebrionis have been used commercially to control certain insectsof the orders diptera and coleoptera, respectively (Gaertner, F. H.,1989, “Cellular Delivery Systems for Insecticidal Proteins: Living andNon-Living Microorganisms,” in Controlled Delivery of Crop ProtectionAgents, R. M. Wilkins, ed., Taylor and Francis, New York and London,1990, pp. 245-255; Couch T. L., 1980, “Mosquito Pathogenicity ofBacillus thuringiensis var. israelensis”, Development in IndustrialMicrobiology 22: 61-76 and Beegle C. C., 1978, “Use of EntomogenousBacteria in Agroecosystems”, “Developments in Industrial Microbiology20: 97-104; may be sited for this). Kreig et. al. (1983) in Z. ang. Ent.96: 500-508, describe Bacillus thuringiensis var. tenebrionis, which isreportedly active against two beetles in the order Coleoptera i.e.,Colorado potato beetles, Leptinotarsa decemlineata and Agelastica alni.

Reference may be made to Crickmore et. al., 1998, “Revision in thenomenclature for the Bacillus thuringiensis pesticidal crystalproteins”, Microbiol. Mol. Biol. Rev. 62: 807-813, wherein crystalprotein genes are classified into 22 classes, primarily on the basis ofamino acid sequence homology. The cloning and expression of a B.t.crystal protein gene in Escherichia coli has been described in severalcases in the published literature (Schnepf et al., 1981, “Cloning andexpression of the Bacillus thuringiensis crystal protein gene inEscherichia coli” may be cited for this). U.S. Pat. Nos. 4,448,885 and4,467,036 disclose the expression of B.t crystal protein in E. coli.U.S. Pat. Nos. 4,797,276 and 4,853,331 disclose B. thuringiensis straintenebrionis, which can be used to control coleopteran pests in variousenvironments. U.S. Pat. No. 4,918,006 discloses B.t. toxins havingactivity against dipterans. U.S. Pat. No. 4,849,217 discloses B.t.isolates, which have activity against the alfalfa weevil. U.S. Pat. No.5,208,017 discloses coelopteran-active Bacillus thuringiensis isolates.U.S. Pat. Nos. 5,151,363 and 4,948,734 disclose certain isolates ofB.t., which have activity against nematodes. Extensive research andresources are being spent to discover new B.t. isolates and their uses.As of now, the discovery of new B.t. isolates and new uses of the knownB.t. isolates remains an empirical, unpredictable art. Severallaboratories all over the world are trying to isolate new δ-endotoxingenes from B. thuringiensis for different host range and mechanism ofaction.

Bulla et al., 1980, “Ultrastructure, physiology and biochemistry ofBacillus thuringiensis”. CRC Crit. Rev. Microbiol. 8: 147-204 andGrochulski et al., 1995, “Bacillus thuringiensis CryIA(a) insecticidaltoxin: crystal structure and channel formation”, J. Mol. Biol, 254:447-464; have reported that majority of B.t. insecticidal crystalproteins are synthesised in natural form as protoxins (molecular weight130-140 kDa), which form parasporal inclusions by virtue of hydrophobicinteractions, hydrogen bondings and disulfide bridges. The protoxins,which are not toxic to insect larvae, are composed of two segments the—N-terminal half and C-terminal half. The protoxins are converted intofunctionally active toxins (60-70 kDa) in insect mid gut following theirsite-specific cleavage by proteases at alkaline pH. Such proteolyticallyprocessed, truncated δ-endotoxins bind to specific receptors in insectmid-gut and cause mortality by making pores in the epithelial membrane(the references, Bietlot et al., 1989, “Facile preparation andcharacterization of the toxin from Bacillus thuringiensis var. kustaki”,Biochem. J., 260: 87-91; Choma et al., 1990, “Unusual proteolysis of theprotoxin and toxin from Bacillus thuringiensis: structuralimplications”, Eur. J. Biochem. 189: 523-27; and Hofte et al., 1986,“Structural and functional analysis of a cloned delta endotoxin of.Bacillus thuringiensis berliner 1715”, Eur. J. Biochem. 161: 273-280;may be cited for this). The protease-resistant active toxin correspondsto N-terminal half of the protoxin molecule. The other segmentcorresponding to C-terminal half is believed to be required for theformation of highly stable crystals. During the proteolytic processing,a small polypeptide comprising about 25-30 amino acid residues isremoved from N-terminal of the protoxin.

The crystal structure of the core toxic segment of CryIAa and Cry3Aaδ-endotoxins are known (Grochulsky et al., 1995, “Bacillus thuringiensisCryIA(a) insecticidal toxin: crystal structure and channel formation”,J. Mol. Biol. 254: 447-464 and Li et al., 1991, “Crystal structure ofinsecticidal δ-endotoxin from Bacillus thuringiensis at 2.5 Åresolution”, Nature. 353: 815-821) and their three-dimensionalstructures are superimposable. Reasonably conserved polypeptide domainssuggest that related toxins have similar topological structure. Theseare globular molecules composed of 3 distinct structural domainsconnected by small peptide linkers. There are no crossovers of thepolypeptide chains between the domains. Domain I consists of 7 α helicalstructures. Domain II consists of three anti-parallel β-sheets and twoshort α-helices. Domain III is a β-sandwich of two anti-parallel highlytwisted β-sheets. Domains II and III are located on the side where theyface helix α7 of domain 1. These domains are closely packed by virtue ofnumerous van der Wall forces, hydrogen bonds and electrostaticinteractions (salt bridges) between the domains.

One of the major reasons for narrow host range of δ-endotoxins is thatthese proteins need specific receptors in the insect gut in order tomake pores and cause toxicity. Since a given δ-endotoxin exhibitstoxicity to a very narrow range of insects, it is desirable to engineerthese proteins for modifying their receptor recognition in larvalmidgut, to widen host range and to improve toxicity. Two approaches havebeen followed for this purpose—first, the development of chimeric (orhybrid) genes by exchanging functional domains of the proteins andsecondly, the development of improved δ-endotoxinproteins by sitedirected mutagenesis. References may be made to U.S. Pat. Nos. 5,128,130and 5,055,294 wherein hybrid B.t. crystal proteins have beenconstructed, which exhibit increased toxicity and display an expandedhost range to the target pests.

The reference may be made to Honee et al., 1990, “A translation fusionproduct of two different insecticidal crystal protein gene of Bacillusthuringiensis exhibits an enlarged insecticidal spectrum” Appl. Environ.Microbiol. 56: 823-825, wherein translational fusion of two cry genes(cry1Ab and cry1Ca) has been made. The resulting hybrid protein hadwider toxicity spectrum that overlapped those of the two contributingparental crystal proteins. However, the drawback is that the activity ofthe chimeric toxin did not increased over any of the parental toxinstowards the target insect pests. In spite of poor toxicity, fusion genewas expressed in tobacco after partial modification, which conferredonly partial protection to transgenic plants against a broader range ofinsects, including Spodoptera exigua, Heliothis virescens and Manducasexta (the reference, van der Salm et al., 1994, “insect resistance oftransgenic plants that express modified Bacillus thuringiensis cryIAband cryIC genes: a resistance management strategy”, Plant Mol. Biol. 26:51-59, may be cited for this).

The reference may be made to Honee et at, 1991, “The C-terminal domainof the toxic fragment of Bacillus thuringiensis crystal proteindetermines receptor binding”, Mol. Microbiol. 5: 2799-2806, wherein 11chimeric genes have been constructed using cry1Ab and cry1Ca as parentgenes by exchanging functional domains. The draw back is that only twochimeric proteins, in which pore-forming domains had been exchanged,exhibited insecticidal activity. However, the efficacy of the toxinchimeric proteins was lower than the parental proteins. Other hybridproteins were non-toxic.

Masson et al., 1992, “Insecticidal properties of a crystal protein geneproduct isolated from Bacillus thuringiensis subsp. kenyae”, Appl.Environ. Microbiol. 58: 2, 642-646, reported that one of the Cry1δ-endotoxins, namely Cry1Ea does not exhibit toxicity against Spodopteralarvae. Further, the reference may be made to Bosch et al., 1994,“Recombinant Bacillus thuringiensis Crystal protein with new properties:possibilities for resistance management”, Bio/Tech 12: 915-918, whereinmany chimeric genes have been developed following in vivo recombinationof cry1Ca and cry1Ea genes. The δ-endotoxin expressed from one of thechimeric genes, which consisted of domain and II of Cry1Ea and domainIII of Cry1Ca protein exhibited larvicidal activity. The transfer ofdomain III of Cry1Ca to Cry1Ea protein gave an insecticidal protein.However, the chimeric toxin was not an improved toxin over the Cry1Ca,which is best-reported toxin to Spodoptera sp. Another chimeric toxinexhibited very poor toxicity. The remaining chimeric toxins were eitherunstable or non-toxic.

Reference may be cited as Rang et al., 1999, “Interaction betweenfunctional domain of Bacillus thuringiensis insecticidal crystalprotein”, Appl Environ Microbial, 65, 7: 2918-25, wherein many chimericgenes have been developed by exchanging the regions coding for eitherdomain 1 or domain III among Cry1Ab, Cry1Ac, Cry1Ca and Cry1Eaδ-endotoxins and checked their stability in E. coli and plasma membranepermeability of Sf9 cells. A chimeric toxin (consisting of domains I andII of Cry1Ca and domain III of Cry1Ab) was more toxic than the parentaltoxins. Exchange of domain III of Cry1Ab with that of Cry1Ca made thechimeric protein more active than the Cry1Ca protein. Proteins with theexchange of other domains were either unstable or less toxic than theparent proteins. However, the toxicity of the chimeric protein to insectlarvae was not tested. Pore formation in insect cell line was comparedbut that cannot be correlated with the insecticidal activity of theδ-endotoxin.

Reference may be made to Chandra et al., 1999, “Amino acid substitutionin alpha-helix 7 of Cry1Ac δ-endotoxin of Bacillus thuringiensis leadsto enhanced toxicity to Helicoverpa armigera Hubner”, FEBS Lett. 458:175-179; wherein a hydrophobic motif in the C-terminal end of thefragment B of diphtheria toxin was found to be homologous to helix α7 ofδ-endotoxins. Upon substitution of helix α7 of Cry1Ac protein by thispolypeptide, the chimeric protein exhibited 7-8 fold enhancement intoxicity towards Helicoverpa armigera. The increased toxicity was due tohigher pore forming ability.

These examples establish the potential of protein engineering for theimprovement of native toxins, to develop commercially usefulδ-endotoxins.

Most of the lepidopteran pests are polyphagous in nature. Spodoptera isa common lepidopteran insect and its 5 species (litura, littoralis,exigua, frugiperda and exempia) are found worldwide. Spodopteralittoralis (the Egyptian cotton leaf worm, CLW) is a major pest ofcotton and other crops of agronomical importance in Europe (thereference Mazier et al., 1997, “The cryIC gene from Bacillusthuringiensis provides protection against Spodoptera littoralis in youngtransgenic plants”, Plant Sci. 127: 179-190, 190, may be cited forthis). It is a notorious pest of cotton, groundnut, chilli, pulses andseveral vegetable crops, especially in warm and humid regions, as thesouthern parts of India. High fecundity, short life cycle, destructivefeeding habits and often-reported emergence of resistance to chemicalinsecticides have made the control of Spodoptera an increasingagricultural problem. Reference may be made to Bai et al., 1993,“Activity of insecticidal proteins and strains of Bacillus thuringiensisagainst Spodoptera exempla (Walker)” J. Inverteb. Pathol. 62: 211-215,wherein it is discussed that the young larvae are susceptible to certainδ-endotoxins, but the larvae beyond 2^(nd) instar display considerabletolerance. This has been attributed to the high content of alkalineproteases in the gut juice (the reference Keller et al., 1996,“Digestion of δ-endotoxin by gut proteases may explain reducedsensitivity of advanced instar larvae of Spodoptera littoralis toCryIC”, Insect Biochem. Mol. Biol. 26: 365-373, may be cites for this).

Four different δ-endotoxins have been reported to cause low level ofmortality to the Spodoptera sp. Of these, Cry1Ca is the most effectivetoxin. The plants expressing Cry1Ca at a high level caused mortality andhence conferred protection against early instar larvae (the referencesMazier et al., 1997, “The cryIC gene from Bacillus thuringiensisprovides protection against Spodoptera littoralis in young transgenicplants”. Plant Sci. 127: 179-190 and Strizhov et al., 1996, “A syntheticcry1C gene, encoding a Bacillus thuringiensis δ-endotoxin, confersSpodoptera resistance in alfalfa and tobacco” Proc. Natl. Acad. Sci.USA. 93: 15012-15017 may be cited for this). However, completeprotection against Spodoptera has not been reported in any case. Thelarvae in advanced developmental stages are not killed at moderatelevels of the known 6-endotoxins. Hence, transgenic plants expressingCry1Ca are not as effective as desirable in protection againstSpodoptera. Other genes, like cry1Cb, cry1Ea and cry1F have not beenemployed for the development of transgenic plants against Spodopterabecause of their comparatively low toxicity. Cry1Cb δ-endotoxin is5-fold less toxic than Cry1Ca. The toxicity of Cry1Ea δ-endotoxin isvery low and is disputed in certain reports (Masson et al., 1992 andBosch et al., 1994 may be cited for this). Cry1F exhibits mild toxicityto Spodoptera larvae (Chambers et al., 1991 may be cited for this).

Reference may be made to Kalman et al., 1993, “Cloning of a novelcryIC-type gene from a strain of Bacillus thuringiensis subsp.Galleriae”, Appl. Environ. Microbiol. 59:4:1131-1137, wherein Cry1Cbδ-endotoxin is reported to be 5-fold less toxic than Cry1Ca. First twodomains of these proteins are highly homologous (92% identical). A majordifference is observed in domain III that exhibits only 48% homology.Higher toxicity (5-fold) of Cry1Ca over Cry1Cb δ-endotoxin suggested usthat domain III of Cry1Ca might have an important role in its efficacy.The toxicity of Cry1Ea δ-endotoxin is very poor as it binds to thereceptor very weakly in the midgut of Spodoptera exigua but the exchangeof Domain III of Cry1Ca with Cry1Ea, made the latter toxic. Thissuggests the role of Domain III of Cry1C protein in receptor binding inthe midgut of Spodoptera (the reference Bosch et. al., 1994,“Recombinant Bacillus thuringiensis Crystal protein with new properties:possibilities for resistance management”. Bio/Tech 12: 915-918, may becited for this). In this publication, Bosch et al. (1994) establishedthe advantage of hybrid toxin as it binds to a receptor where Cry1Cadoes not bind. However, the toxicity of both, the native Cry1Ca and thehybrid Cry1Ea was comparable. They filed a patent (U.S. Pat. No.5,736,131) in which 1.9-fold improvement in the toxicity towardsSpodoptera exigua was claimed. The difference in results in thepublication that reports no enhancement in toxicity and in the patentthat claims 1.9 fold improved toxicity, makes the overall pictureunclear.

Plant genetic engineering technology has made significant progressduring the last 10 years. It has become possible to stably introduceforeign genes into plants. This has provided exciting opportunities formodern agriculture. Derivatives of the Ti-plasmid of the plant pathogen,Agrobacterium tumefaciens, have proven to be efficient and highlyversatile vehicles for the introduction of foreign genes into planttissue. In addition, a variety of methods to deliver DNA, such aselectroporation, microinjection, pollen-mediated gene transfer andparticle gun technology, have been developed for the same purpose.

The major aim of plant transformation by genetic engineering has beencrop improvement. A substantial effort has been made for engineering theplants for useful traits such as insect-resistance. In this respect,progress in engineering insect resistance in transgenic plants has beenachieved through the use of genes, encoding δ-endotoxins, from B.thuringiensis strains. Since δ-endotoxins possess a specificinsecticidal spectrum and display no toxicity towards other non-hostanimals and humans, these are highly suited for developing commerciallyuseful plants. No other protein is known which shows as high toxicity as(at ppm levels) and is still as safe to non-target organisms as theδ-endotoxins.

The feasibility of generating insect-resistant transgenic cropsexpressing 8-endotoxins and their success in commercial agriculture hasbeen demonstrated. (References may be made to Vaeck et al., 1987,“Transgenic plants protected from insect attack”, Nature. 328: 33-37;Fischoff et al., 1987, “Insect tolerant transgenic tomato plants”,Bio/Tech. 5: 807-813″; Barton et al., 1987, “Bacillus thuringiensisδ-endotoxin expressed in transgenic Nicotiana tabaccum providesresistance to lepidopteran insects”, Plant Physiol. 85: 1103-1109 may bemade for this). Transgenic plants offer an attractive alternative toinsect control in agriculture, which is at the same time safe,environment friendly and cost-effective. Successful insect control hasbeen observed under field conditions (Reference may be made to Delannayet al., 1989; Meeusen and Warren, 1989).

A reference may be cited to Von Tersch et al. 1991, “Insecticidal toxinsfrom Bacillus thuringiensis subsp kanyae: Gene cloning andcharacterization and comparison with B. thuringiensis subsp kurstakiCry1A(c) toxin” in Appl and Environ Microbial, 57: 2: 349-58, whereintwo variants of cry1Ac were isolated from two different strains. Theiramino acid composition was different at 7 positions. Both theδ-endotoxins were expressed in E. coli and toxicity experiment wasconducted. The two toxins did not exhibit any difference in efficacytowards target pests.

In another study (Schnepf et al., 1998, “Bacillus thuringiensis and itspesticidal crystal proteins”, 62: 3, 775-806), amino acid residues GYYof Cry1Ac δ-endotoxin at position 312 to 314 were altered to replacethese with ASY, GSY and GFS. No difference in toxicity of the threeproteins was noticed.

There are two natural variants of Cry1C δ-endotoxin namely Cry1Ca andCry1Cb. These proteins show 81% (Schnepf et al., 1998, “Bacillusthuringiensis and its pesticidal crystal proteins”, 62: 3, 775-806)amino acid sequence identity. Despite this difference, both the toxinsare toxic to their target pest, though there is some difference in thelevel of toxicity. Their host range is also same. (Kalman et al., 1993.“Cloning of a noval cry1C type gene from a strain of Bacillusthuringiensis subsp galleriae” Appl. Environ Microbial 59: 4: 1131-37)

OBJECTS OF THE PRESENT INVENTION

The main object of the present invention is to develop a chimeric δendotoxin protein.

Another main object of the present invention is to develop a chimeragene coding for a chimeric δ endotoxin protein.

Yet another object of the present invention is to develop a method ofdeveloping said chimeric protein.

Still another object of the present invention is to develop a method ofoverexpressing said chimeric protein in a suitable microbe.

Still another object of the present invention is to develop a method oftreating insect infested plants using said chimeric protein.

Further object of the present invention is to develop an insecticide formultidrug resistant insects.

Another object of the present invention is to develop an effectiveinsecticide.

Yet another object of the present invention is to develop an insecticidehaving no adverse effect on the plants.

Still another object of the present invention is to develop aninsecticide with about 100% insecticidal activity.

SUMMARY OF THE PRESENT INVENTION

The present invention relates to a chimeric δ endotoxin protein Cry 1Eof SEQ ID No. 1 with extraordinarily high insecticidal property and achimera gene of SEQ ID No. 2 to encoding the said chimeric protein, andalso a method of treating insect infested plants using said chimeraprotein.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

Accordingly, the present invention relates to a chimeric δ endotoxinprotein Cry 1E of SEQ ID No. 1 with extraordinarily high insecticidalproperty and a chimera gene of SEQ ID No. 2 encoding the said chimericprotein, and also a method of treating insect infested plants using saidchimera protein.

In one embodiment of the present invention, a chimeric δ endotoxinprotein Cry1E of SEQ ID No. 1 and other proteins of 75% and abovehomology in the sequence.

In another embodiment of the present invention, a chimeric δ endotoxinprotein Cry1E of SEQ ID No. 1.

In yet another embodiment of the present invention, wherein said proteinis of length 641 amino acid residues.

In still another embodiment of the present invention, wherein saidchimeric protein is designed from δ endotoxins Cry1Ea and Cry1Ca ofBacillus thuringiensis.

In still another embodiment of the present invention, wherein saidchimeric protein of length of 641 residues is consisting of residues 1to 529 from endotoxin Cry1Ea of same position, residues 530 to 599 fromCry1Ca of position 533 to 602, residues 600 to 616 from Cry1Ea ofposition 588 to 604 and residues 617 to 641 of a synthetic polypeptide.

In still another embodiment of the present invention, wherein peptidedomain from 530 to 587 of Cry1Ea can be replaced with that of any otherδ endotoxin.

In still another embodiment of the present invention, wherein last 25amino acid residues improve the stability against the proteases ofplants.

In still another embodiment of the present invention, wherein saidchimeric protein is stable at temperature ranging between 4-35° C.

In further embodiment of the present invention, A chimera gene of SEQ IDNo. 2.

In another embodiment of the present invention, wherein said chimeraencodes chimeric protein of SEQ ID No. 1.

In still another embodiment of the present invention, wherein saidchimera is of length 1990 base pairs (bp).

In still another embodiment of the present invention, wherein saidchimera is a 1.99-kb double stranded DNA.

In still another embodiment of the present invention, wherein saidchimera contains plant preferred codon distributed evenly to facilitateefficient translation.

In still another embodiment of the present invention, wherein saidchimera contains plant preferred translation initiation codon of ATGGCTat 5′ extreme.

In still another embodiment of the present invention, wherein saidchimera contains plant preferred translation termination codon ofTAATGA.

In still another embodiment of the present invention, wherein saidchimera contains 33 restriction sites distributed uniformly throughoutthe length of the gene at a distance of about 40-80 bp.

In still another embodiment of the present invention, whereinrestriction sites are enzymes selected from a group comprising Hind III,EcoRI, and BamHI.

In still another embodiment of the present invention, wherein saidchimera is divided into 58 overlapping oligonucleotides of length 40 to65 by each located at a distance of 6 to 26 base pairs. (bp)

In still another embodiment of the present invention, wherein saidchimera contains said overlapping nucleotides with an overlap of 13 to18 nucleotides with the immediately adjacent oligonucleotides on thecomplementary strain.

In still another embodiment of the present invention, wherein saidchimera has T_(m) value ranging between 44 to 55° C.

In further embodiment of the present invention, a method ofoverexpressing insecticidal chimeric protein Cry1E in microbes.

In another embodiment of the present invention, cloning gene Cry1E ofSEQ ID No. 2 encoding said chimeric protein in a vector.

In still another embodiment of the present invention, transformingmicrobe with said cloned vector.

In still another embodiment of the present invention, overexpressingsaid chimeric protein into said microbe.

In still another embodiment of the present invention, wherein saidchimera is expressed into a microbe selected from a group comprisingbacteria, algae, and fungi.

In still another embodiment of the present invention, whereinrestriction enzymes for said cloning are selected from a groupcomprising Hind III, EcoRI, Ncol, Mfe I and BamHI.

In still another embodiment of the present invention, wherein inducingoverexpression of said protein by using isopropylthiogalactoside (IPTG).

In still another embodiment of the present invention, whereinoverexpressing said protein at 15° C. to avoid mis-folding of saidproteins.

In still another embodiment of the present invention, wherein saidvectors are selected from a group comprising Plasmids, viral DNA, andcosmids.

In still another embodiment of the present invention, wherein expressionof chimera in the microbe is confirmed by RT-PCR, western Analysis, andELISA.

In still another embodiment of the present invention, wherein presenceof chimera in the microbe is confirmed by PCR and southern Analysis.

In further embodiment of the present invention, a method of treatingplants infected with insects using said insecticidal chimeric protein.

In another embodiment of the present invention, incorporating geneencoding chimera protein Cry1E into plant infected with insects.

In yet another embodiment of the present invention, exposing transgenicplant to insects.

In still another embodiment of the present invention, determininginsecticidal activity of said transgenic plants.

In still another embodiment of the present invention, wherein insectpests are selected from a group comprising spodoptera sp., andHelicoverpa sp.

In still another embodiment of the present invention, wherein plants areselected from a group comprising tobacco, cotton, chickpea, pegeonpea,groundnut, cauliflower, cabbage, chilli, and capsicum.

In still another embodiment of the present invention, whereinrestriction enzymes for said cloning are selected from a groupcomprising Hind III, EcoRI, Ncol, and BamHI.

In still another embodiment of the present invention, wherein chimericprotein shows high degree of expression in plants by having about 0.5%of total soluble protein of plants.

In still another embodiment of the present invention, wherein saidchimeric protein is stable in said transgenic plant.

In still another embodiment of the present invention, wherein insectsexposed to said chimeric protein show weight loss before death.

In still another embodiment of the present invention, wherein saidchimeric protein shows insecticidal property against insect at alldevelopmental stages.

In still another embodiment of the present invention, wherein saidchimeric protein is multifold more potent insecticide as compared toparental proteins.

In still another embodiment of the present invention, whereininsecticidal activity of said chimeric protein shows mortality of insectpests ranging between 80-100% within about 4 hours of exposure.

In still another embodiment of the present invention, wherein insectsexposed to said chimeric protein for about 1 hour shows delayeddevelopment, infertility and disrupted metamorphosis.

In still another embodiment of the present invention, wherein EC₅₀ forHelicoverpa sp. is ranging between 250-350 ng/ml of artificial diet ofinsects.

In still another embodiment of the present invention, wherein EC₅₀ ofSpodoptera sp. is ranging between 25-50 ng/ml of artificial diet.

In still another embodiment of the present invention, the said methodshows no adverse effect on the normal growth of the transformed plants.

In further embodiment of the present invention, a novel chimericBacillus thuringiensis δ-endotoxin is strategically developed byreplacing a polypeptide domain of a δ-endotoxin, herein said Cry1Ea withthe corresponding domain of other δ-endotoxins and a novel polypeptideat the C-terminus extreme. A gene is theoretically designed andchemically synthesized to encode the novel chimeric toxin and express itat high level in plant tissue. The gene was expressed in a microbe (E.coli) and two dicot plants (tobacco and cotton). Efficacy of thestrategically designed δ-endotoxin was established in both the systems.The toxicity of the chimeric protein to lepidopteran insect larvae(Spodoptera and Helicoverpa) was improved as compared to the parentproteins. The chimeric synthetic gene is commercially valuable as it canbe used to develop agronomically improved crop plants for resistance toinsect pests.

In another embodiment of the present invention, accordingly, the presentinvention provides “a novel δ-endotoxin improved for insecticidalactivity and a gene for its high level expression in plants” whichcomprises strategic designing of a novel chimeric δ-endotoxin, hereinsaid chimeric Cry1E (616 amino acid residues), by replacing apolypeptide domain (from position 530 to 587) of Cry1Ea protein by thatof Cry1Ca (from position 533 to 602), incorporation of a novelpolypeptide of 25 amino acid residues at the C-terminus extreme,theoretical designing of a gene to code 641 amino acid residue longchimeric δ-endotoxin at a high level in plants, designing and chemicalsynthesis of oligonucleotides representing theoretically designed gene,assembly of oligonucleotides into double stranded DNA, cloning andsequence analysis of cloned synthetic DNA, construction of vectors forthe expression of chimeric gene in E. coli and plants, expression ofsynthetic chimeric gene in E. coli, comparison of the toxicity of thechimeric protein with the parental proteins against Spodoptera lituraand Helicoverpa armigera, transformation of tobacco with the chimericgene, high level expression of the engineered protein in transgenicplants, purification of chimeric δ-endotoxin protein from transgenicplants and confirmation of its efficacy on larvae of Spodoptera lituraand Helicoverpa armigera, evaluation of the potential of the chimerictoxin expressed in transgenic plants for protection against targetinsect pests.

In an embodiment of the present invention, a naturally occurringδ-endotoxin, namely Cry1Ea has been strategically designed and modifiedaccordingly to make it biologically active against larvae of insectpests, like Spodoptera sp.

In another embodiment of the present invention, 616 amino acid residuelong chimeric δ-endotoxin, herein said chimeric Cry1E, was theoreticallydesigned by replacing a peptide domain of Cry1Ea from position 530 to587 by that of Cry1Ca from position 533 to 602. Further, a novelpolypeptide of 25 amino acid residues was introduced at the C-terminusextreme of δ-endotoxin.

In yet another embodiment of the present invention, two peptide domainsfrom positions 530 to 545 and 578 to 587 of Cry1Ea can be replaced bythose of Cry1Ca from 533 to 555 and 588 to 602. Such chimeric toxin mayalso perform similar (or equivalent) biological activity.

In still another embodiment of the present invention, the peptide domainfrom 530 to 587 of Cry1Ea can be replaced by that of other δ-endotoxins.

In still another embodiment of the present invention, a polypeptide of25 amino acid residues is introduced at the C-terminus of δ-endotoxin.This polypeptide improved the stability of δ-endotoxin against theproteases of plant. This might have improved the stability of theδ-endotoxin insect mid gut.

In still another embodiment of the present invention, 25 amino acidresidues long polypeptide may be included at the C-terminus of otherδ-endotoxins, such toxins may become stable against different kind ofproteases.

In still another embodiment of present invention, 25 amino acid residueslong polypeptide may be incorporated at the C-terminus of anyrecombinant protein for their stability against proteases.

In still another embodiment of the present invention, a 1.99-kb doublestranded DNA was theoretically designed to encode the chimeric protein.Plant preferred codons were used to encode amino acids of the chimerictoxin protein to facilitate high-level expression of the gene in plants.

There are 6 variants of Cry1Aa δ-endotoxin namely Cry1Aa1 to Cry1Aa6given in EMBL database. Clustal analysis for amino acid sequence ofthese δ-endotoxin proteins is shown below. At three positions (77, 148and 302), the amino acid residues are different. The variant positionsare shown below in bold letters. However, all six genes have beendeployed in toxicity experiments by different laboratories. The efficacyof all six variants in their toxicity towards insects is comparable.

CRY1AA1 MDNNPNINECIPYNCLSNPEVEVLGGERIETGYTPIDISLSLTQFLL  50 SEF CRY1AA4MDNNPNINECIPYNCLSNPEVEVLGGERIETGYTPIDISLSLTQFLL  50 SEF CRY1AA5MDNNPNINECIPYNCLSNPEVEVLGGERIETGYTPIDISLSLTQFLL  50 SEF CRY1AA6MDNNPNINECIPYNCLSNPEVEVLGGERIETGYTPIDISLSLTQFLL  50 SEF CRY1AA2MDNNPNINECIPYNCLSNPEVEVLGGERIETGYTPIDISLSLTQFLL  50 SEF CRY1AA3MDNNPNINECIPYNCLSNPEVEVLGGERIETGYTPIDISLSLTQFLL  50 SEF CRY1AA1VPGAGFVLGLVDIIWGIFGPSQWDAFPVQIEQLINQRIEEFARNQAI 100 SRL CRY1AA4VPGAGFVLGLVDIIWGIFGPSQWDAFPVQIEQLINQRIEEFARNQAI 100 SRL CRY1AA5VPGAGFVLGLVDIIWGIFGPSQWDAFLVQIEQLINQRIEEFARNQAI 100 SRL CRY1AA6VPGAGFVLGLVDIIWGIFGPSQWDAFLVQIEQLINQRIEEFARNQAI 100 SRL CRY1AA2VPGAGFVLGLVDIIWGIFGPSQWDAFLVQIEQLINQRIEEFARNQAI 100 SRL CRY1AA3VPGAGFVLGLVDIIWGIFGPSQWDAFLVQIEQLINQRIEEFARNQAI 100 SRL CRY1AA1EGLSNLYQIYAESFREWEADPTNPALREEMRIQFNDMNSALTTAIPL 150 LAV CRY1AA4EGLSNLYQIYAESFREWEADPTNPALREEMRIQFNDMNSALTTAIPL 150 LAV CRY1AA5EGLSNLYQIYAESFREWEADPTNPALREEMRIQFNDMNSALTTAIPL 150 LAV CRY1AA6EGLSNLYQIYAESFREWEADPTNPALREEMRIQFNDMNSALTTAIPL 150 LAV CRY1AA2EGLSNLYQIYAESFREWEADPTNPALREEMRIQFNDMNSALTTAIPL 150 FAV CRY1AA3EGLSNLYQIYAESFREWEADPTNPALREEMRIQFNDMNSALTTAIPL 150 FAV CRY1AA1QNYQVPLLSVYVQAANLHLSVLRDVSVFGQRWGFDAATINSRYNDLT 200 RLI CRY1AA4QNYQVPLLSVYVQAANLHLSVLRDVSVFGQRWGFDAATINSRYNDLT 200 RLI CRY1AA5QNYQVPLLSVYVQAANLHLSVLRDVSVFGQRWGFDAATINSRYNDLT 200 RLI CRY1AA6QNYQVPLLSVYVQAANLHLSVLRDVSVFGQRWGFDAATINSRYNDLT 200 RLI CRY1AA2QNYQVPLLSVYVQAANLHLSVLRDVSVFGQRWGFDAATINSRYNDLT 200 RLI CRY1AA3QNYQVPLLSVYVQAANLHLSVLRDVSVPGQRWGFDAATINSRYNDLT 200 RLI CRY1AA1GNYTDYAVRWYNTGLERVWGPDSRDWVRYNQFRRELTLTVLDIVALF 250 SNY CRY1AA4GNYTDYAVRWYNTGLERVWGPDSRDWVRYNQFRRELTLTVLDIVALF 250 SNY CRY1AA5GNYTDYAVRWYNTGLERVWGPDSRDWVRYNQFRRELTLTVLDIVALF 250 SNY CRY1AA6GNYTDYAVRWYNTGLERVWGPDSRDWVRYNQFRRELTLTVLDIVALF 250 SNY CRY1AA2GNYTDYAVRWYNTGLERVWGPDSRDWVRYNQFRRELTLTVLDIVALF 250 SNY CRY1AA3GNYTDYAVRWYNTGLERVWGPDSRDWVRYNQFRRELTLTVLDIVALF 250 SNY CRY1AA1DSRRYPIRTVSQLTREIYTNPVLENFDGSFRGMAQRIEQNIRQPHLM 300 DIL CRY1AA4DSRRYPIRTVSQLTREIYTNPVLENFDGSFRGMAQRIEQNIRQPHLM 300 DIL CRY1AA5DSRRYPIRTVSQLTREIYTNPVLENFDGSFRGMAQRIEQNIRQPHLM 300 DIL CRY1AA6DSRRYPIRTVSQLTREIYTNPVLENFDGSFRGMAQRIEQNIRQPHLM 300 DIL CRY1AA2DSRRYPIRTVSQLTREIYTNPVLENFDGSFRGMAQRIEQNIRQPHLM 300 DIL CRY1AA3DSRRYPIRTVSQLTREIYTNPVLENFDGSFRGMAQRIEQNIRQPHLM 300 DIL CRY1AA1NSITIYTDVHRGFNYWSGHQITASPVGFSGPEFAFPLFGNAGNAAPP 350 VLV CRY1AA4NSITIYTDVHRGFNYWSGHQITASPVGFSGPEFAFPLFGNAGNAAPP 350 VLV CRY1AA5NSITIYTDVHRGFNYWSGHQITASPVGFSGPEFAFPLFGNAGNAAPP 350 VLV CRY1AA6NSITIYTDVHRGFNYWSGHQITASPVGFSGPEFAFPLFGNAGNAAPP 350 VLV CRY1AA2NRITIYTDVHRGFNYWSGHQITASPVGFSGPEFAFPLFGNAGNAAPP 350 VLV CRY1AA3NSITIYTDVHRGFNYWSGHQITASPVGFSGPEFAFPLFGNAGNAAPP 350 VLV CRY1AA1SLTGLGIFRTLSSPLYRRIILGSGPNNQELFVLDGTEFSFASLTTNL 400 PST CRY1AA4SLTGLGIFRTLSSPLYRRIILGSGPNNQELFVLDGTEFSFASLTTNL 400 PST CRY1AA5SLTGLGIFRTLSSPLYRRIILGSGPNNQELFVLDGTEFSFASLTTNL 400 PST CRY1AA6SLTGLGIFRTLSSPLYRRIILGSGPNNQELFVLDGTEFSFASLTTNL 400 PST CRY1AA2SLTGLGIFRTLSSPLYRRIILGSGPNNQELFVLDGTEFSFASLTTNL 400 PST CRY1AA3SLTGLGIFRTLSSPLYRRIILGSGPNNQELFVLDGTEFSFASLTTNL 400 PST CRY1AA1IYRQRGTVDSLDVIPPQDNSVPPRAGFSHRLSHVTMLSQAAGAVYTL 450 RAP CRY1AA4IYRQRGTVDSLDVIPPQDNSVPPRAGFSHRLSHVTMLSQAAGAVYTL 450 RAP CRY1AA5IYRQRGTVDSLDVIPPQDNSVPPRAGFSHRLSHVTMLSQAAGAVYTL 450 RAP CRY1AA6IYRQRGTVDSLDVIPPQDNSVPPRAGFSHRLSHVTMLSQAAGAVYTL 450 RAP CRY1AA2IYRQRGTVDSLDVIPPQDNSVPPRAGFSHRLSHVTMLSQAAGAVYTL 450 RAP CRY1AA3IYRQRGTVDSLDVIPPQDNSVPPRAGFSHRLSHVTMLSQAAGAVYTL 450 RAP CRY1AA1TFSWQHRSAEFNNIIPSSQITQIPLTKSTNLGSGTSVVKGPGFTGGD 500 ILR CRY1AA4TFSWQHRSAEFNNIIPSSQITQIPLTKSTNLGSGTSVVKGPGFTGGD 500 ILR CRY1AA5TFSWQHRSAEFNNIIPSSQITQIPLTKSTNLGSGTSVVKGPGFTGGD 500 ILR CRY1AA6TFSWQHRSAEFNNIIPSSQITQIPLTKSTNLGSGTSVVKGPGFTGGD 500 ILR CRY1AA2TFSWQHRSAEFNNIIPSSQITQIPLTKSTNLGSGTSVVKGPGFTGGD 500 ILR CRY1AA3TFSWQHRSAEFNNIIPSSQITQIPLTKSTNLGSGTSVVKGPGFTGGD 500 ILR CRY1AA1RTSPGQISTLRVNITAPLSQRYRVRIRYASTTNLQFHTSIDGRPINQ 550 GNF CRY1AA4RTSPGQISTLRVNITAPLSQRYRVRIRYASTTNLQFHTSIDGRPINQ 550 GNF CRY1AA5RTSPGQISTLRVNITAPLSQRYRVRIRYASTTNLQFHTSIDGRPINQ 550 GNF CRY1AA6RTSPGQISTLRVNITAPLSQRYRVRIRYASTTNLQFHTSIDGRPINQ 550 GNF CRY1AA2RTSPGQISTLRVNITAPLSQRYRVRIRYASTTNLQFHTSIDGRPINQ 550 GNF CRY1AA3RTSPGQISTLRVNITAPLSQRYRVRIRYASTTNLQFHTSIDGRPINQ 550 GNF CRY1AAISATMSSGSNLQSGSFRTVGFTTPFNFSNGSSVFTLSAHVFNSGNEVY 600 IDR CRY1AA4SATMSSGSNLQSGSFRTVGFTTPFNFSNGSSVFTLSAHVFNSGNEVY 600 IDR CRY1AA5SATMSSGSNLQSGSFRTVGFTTPFNFSNGSSVFTLSAHVFNSGNEVY 600 IDR CRY1AA6SATMSSGSNLQSGSFRTVGFTTPFNFSNGSSVFTLSAHVFNSGNEVY 600 IDR CRY1AA2SATMSSGSNLQSGSFRTVCFTTPFNFSNGSSVFTLSAHVFNSGNEVY 600 IDR CRY1AA3SATMSSGSNLQSGSFRTVCFFFPFNFSNGSSVFTLSAHVFNSGNEVY 600 IDR CRY1AA1IEFVPAEVT 609 (SEQ ID NO: 3) CRY1AA4 IEFVPAEVT 609 (SEQ ID NO: 4)CRY1AA5 IEFVPAEVT 609 (SEQ ID NO: 5) CRY1AA6 IEFVPAEVT 609 (SEQ ID NO:6) CRY1AA2 IEFVPAEVT 609 (SEQ ID NO: 7) CRY1AA3 IEFVPAEVT 609 (SEQ IDNO: 8)

A reference may be cited to Von Tersch et al. 1991, “Insecticidal toxinsfrom Bacillus thuringiensis subsp kanyae: Gene cloning andcharacterization and comparison with B. thuringiensis subsp kurstakiCry1A(c) toxin” in Appl and Environ Microbial, 57: 2: 349-58, whereintwo variants of cry1Ac were isolated from two different strains. Theiramino acid composition was different at 7 positions. Both theδ-endotoxins were expressed in E. coli and toxicity experiment wasconducted. The two toxins did not exhibit any difference in efficacytowards target pests.

Further, this clearly states that in proteins, more particularly in thefield of endotoxins, the high homology of the sequence is not found tomake any significant difference in activity. The above-referred exampleof endotoxins Cry1Aa1 to Cry1Aa6 clearly reflect the essence of thiswork. In the instant Application, the applicant has observedextraordinarily high insecticidal activity. Further, the homology of 70%and above in the sequence of chimeric protein Cry1E of the instantApplication is also found to show no significant change in the activity.This means that the proteins with sequence homology of 70% and above forchimeric protein Cry 1E are used as insecticidal agents.

In another study (Schnepf et al., 1998, “Bacillus thuringiensis and itspesticidal crystal proteins”, 62: 3, 775-806), amino acid residues GYYof Cry1Ac δ-endotoxin at position 312 to 314 were altered to replacethese with ASY, GSY and GFS. No difference in toxicity of the threeproteins was noticed.

In addition, there are two natural variants of Cry1C δ-endotoxin namelyCry1Ca and Cry1Cb. These proteins show 81% (Schnepf et al., 1998,“Bacillus thuringiensis and its pesticidal crystal proteins”, 62: 3,775-806) amino acid sequence identity. Despite this difference, both thetoxins are toxic to their target pest, though there is some differencein the level of toxicity. Their host range is also same. (Kalman et al.,1993. “Cloning of a noval cry1C type gene from a strain of Bacillusthuringiensis subsp galleriae “Appl. Environ Microbial 59: 4: 1131-37)

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1 shows represents PCR based assembly of 58 overlappingoligonucleotides into 1.99 kb novel chimeric cry1E DNA. Lanes 2-4represent gene assembly in different PCR conditions, the desired DNAfragment is shown with arrow Lane 1 showing λDNA/HindIII-EcoRI molecularweight marker.

FIG. 2 represents the restriction analysis of plasmid pPK59 havingE-35-S promoter at the upstream of the novel chimeric gene. The plasmidwas digested with SalI restriction enzyme (40 by downstream of gene).The linear plasmid was further digested with NcoI (lane 2), NheI (lane3), BstXI (lane 4), NruI (Lane 5) and SacI (lane 6). Lane 1 and 7represent λDNA/HindIII-EcoRI and λDNA/HindIII molecular weight markers,respectively.

FIG. 3 represent map of plasmids pPK202, pPK141, pPK135 and pPk206.

FIG. 4 represents insect bioassay with transgenic tobacco plants. 1^(st)instar larvae of Spodoptera litura showing 100% mortality after 2 daysof feeding on transgenic leaf expressing the novel chimeric cry1E gene(left). Control leaf was eaten by larvae voraciously (right).

FIG. 5 represents 3^(rd) instar larvae of Spodoptera litura showing 100%mortality after 2 days of feeding (left). Transgenic plant leafexhibited high level of protection against larvae in contrast to thecontrol plant leaf (right).

FIG. 6 represents 100% mortality of 5^(th) instar larvae of Spodopterafollowing 48 h of feeding on transgenic tobacco leaf (left). During sameperiod, the larvae fed on control plant leaf ingested 6 leaves (right).

In still another embodiment of the present invention, 58oligonucleotides were designed to represent 1.99-kb chimeric cry1E DNA.All oligonucleotides were synthesised chemically and fused enzymaticallyto obtain desired double stranded DNA. (Please Refer FIG. 1)

In still another embodiment of the present invention, two independentconstructs were made, each for E. coli and plant expression. Theparental genes (cry1Ea and cry1Ca) were also introduced in otherexpression vectors for their expression in E. coli.

In still another embodiment of the present invention, all three geneswere expressed in E. coli. The efficiency of the chimeric Cry1E againstSpodoptera litura was compared to the parental toxins (Cry1Ea andCry1Ca). The toxicity experiments established that the engineered toxinis several fold more toxic as compared to the parental proteins.

In still another embodiment of the present invention, the chimericprotein expressed in plants was shown to be also toxic to Helicoverpaarmigera, another serious insect pest. This established the improvementin the host range of the novel chimeric toxin designed in this study.

In still another embodiment of the present invention, the synthetic genewas introduced in tobacco for expression of the chimeric toxin. Thetransgenic plants expressed chimeric toxin and accumulated up to 0.5% oftotal soluble protein. The transgenic plants exhibited excellentprotection against larvae of Spodoptera litura and caused 100% mortalityat all the developmental stages.

In still another embodiment of the present invention, the novel chimerictoxin was purified from total soluble protein from the leaf of thetransgenic tobacco plant and mixed in semi synthetic diet. The toxicityexperiments again established the efficacy of the hybrid toxin.

The subject invention concerns the discovery of highly active chimericδ-endotoxins. A novel δ-endotoxin, 641 amino acid residues long, hereinsaid chimeric Cry1E was strategically designed by replacing dpolypeptide domain (from position 530 to 587) of Cry1Ea protein by thatof Cry1Ca (from position 533 to 602). In this way chimeric toxincomprises amino acid residues 1-529 of Cry1Ea, 530 to 599 of Cry1Ca and600 to 616 of Cry1Ea. A novel polypeptide of 25 amino acid residues wasincluded as the C-terminus extreme of the δ-endotoxin. In other words,this polypeptide constituted amino acid residue 617-641 of the chimerictoxin. Several chimeric toxins can be created by replacing differentparts of Cry1Ea toxin with strategically designed amino acid sequencesor parts of the other toxins. A 1.99 kb nucleotide sequence wastheoretically designed to code for the above-mentioned chimericδ-endotoxin. The gene encoding toxin protein, herein said chimeric cry1Ewas designed for high-level expression in plants, by introducingplant-preferred codons. The plant preferred codons for each amino acidwere distributed evenly to facilitate efficient translation. Atranslation initiation context appropriate to gene expression in plants(TAAACCATGGCT; (SEQ ID NO:9)) was included at 5′ extreme and twotranslation stop codons (signals) were introduced at the end of thereading frame of the chimeric toxin. A total of 33 unique restrictionsites were introduced uniformly throughout the length of the gene at adistance of 40-80 bp. BamHI and HindIII restriction sites were createdat the upstream and BamHI and EcoRI at the downstream of the gene tofacilitate its cloning. (Please refer FIG. 2).

The translation initiation context automatically created an NcoI site atthe immediate start of the gene. The gene was divided into 58overlapping oligonucleotides (40 to 65 nucleotides long) with 6 to 26base long gaps in between (Please refer FIG. 1). Each oligonucleotidehad 13-18 nucleotide long overlap with the immediately adjacentoligonucleotides on the complementary strand. The complementary overlapswere designed to keep T_(m) value between 48-50° C. The oligonucleotideswere synthesised on a DNA synthesiser (Gene Assembler Special,Pharmacia, Sweden) at 200 nmole scale and purified on denaturingurea-PAGE. All 58 oligonucleotides were assembled into 1.99 kbdouble-stranded DNA, herein said chimeric cry1E gene following theligation-free gene synthesis method of Singh et al. (1996) and as shownin FIG. 1. The DNA was digested with HindIII and EcoRI restrictionenzymes and cloned in pBluescriptll SK(+) (Stratagene, La Jolla,Calif.). The plasmid was named as pPK200. The nucleotide sequence of thesynthetic DNA was confirmed by sequencing the cloned synthetic DNA onautomated DNA sequencing system (Applied Biosystems model 373A).

A cassette was constructed also for the expression of the chimeric toxinin E. coli under the control of T7lac promoter. The plasmid pPK200 wasdigested with the restriction enzymes NcoI and BamHI and cloned inexpression vector pET-19b (Novagen, Madison Wis.). The plasmid was namedas pPK206. DNA encoding Cry1Ca and Cry1Ea toxins were amplified withpolymerase chain reaction, using suitable primers, which created NcoIand BamHI restriction sites at the upstream and the downstream of theamplicon. The amplified products were cloned in the pET-19b vector. Theconstructs having Cry1Ca and Cry1Ea toxin coding DNA were named aspPK135 and pPK141, respectively. E. coli BL21DE3 strain was transformedwith the constructs pPK206, pPK135 and pPK141. (Please refer FIG. 3).The toxin proteins were expressed by induction with appropriateconcentrations of IPTG. The expression was carried out at 15° C. toavoid possible mis-folding. E. coli cells were lysed with lysozyme andsonicated to release the δ-endotoxins. The toxin proteins were found ininclusion bodies. These were solubilised in 50 mM Bicarbonate buffer (pH9.5) at 28° C. The toxin proteins were quantified densitometrically.Serial dilutions of the toxins were mixed in semi-synthetic diet and themixture was poured in the petri dishes. Total E. coli protein served ascontrol diet. Fifteen neonatal larvae of Spodoptera litura were releasedonto the cakes of the diet mixture in a 100-ml beaker and the mouth wascovered with muslin cloth to allow gas exchange. Each experiment wasconducted with 6 replicates. The diet was changed after every alternateday. Bio-assay was conducted with 16/8 h photoperiod at 25±0.2° C.Toxicity data was recorded after 7 days of the feeding. EC₅₀ wasdetermined by standard log-probit analysis. All three proteins weretested simultaneously. The representative results are as shown in Table1 here below.

TABLE 1 δ-endotoxins(S) EC₅₀ (μg/ml semisynthetic diet) Cry1Ea >108Cry1Ca 29.48 ± 1.77 Chimeric Cry1E  6.27 ± 0.59

The result showed that the E. coli strain expressing the chimeric toxinwas several fold more toxic over Cry1Ea and more than four fold toxicover Cry1Ca protein. The Cry1Ea toxin protein failed to cause any effecton the Spodoptera larvae. The result established the successfulengineering of the Cry1Ea toxin to develop a novel protein chimericCry1E, which is biologically active and an improved toxin. Theengineered protein was four fold more toxic than Cry1Ca protein, whichis the best-known 8-endotoxin against Spodoptera sp.

All three δ-endotoxins were also tested against 72-h old larvae ofHelicoverpa sp. Each larva was released on diet in a separate box. 40larvae were challenged with each concentration of the toxin. Thetoxicity results show that the chimeric toxin is also toxic toHelicoverpa. A representative result is shown in Table 2 as here below.

TABLE 2 δ-endotoxins(S) EC₅₀ (μg/ml semisynthetic diet) Cry1Ea >176Cry1Ca 136.22 ± 8.77 Chimeric Cry1E  26.71 ± 1.39

As used here, reference to the “toxin” means, the N-terminal segment,which is responsible for the insect pesticidal activity. A personskilled in this art can convert this toxin into a protoxin by includinga part or complete C-terminus fragment of a homologous or hetrologousδ-endotoxin. Such protoxin will be a very stable molecule inside amicrobial cells for example, in E. coli, Pseudomonas etc. and can beused in developing microbial formulations. Such formulations can be usedas pesticides. Development of such protoxins and formulations using thenovel toxin developed by us is also within the scope of the inventionclaimed by us.

The gene and toxin useful according to the subject invention include notonly 641 amino acid long toxin but also fragments of the novel sequence,variants and mutants, which retain the characteristic pesticidalactivity of the toxin specifically exemplified herein. As used here, theterms “variants” or “variations” of genes refer to nucleotide sequences,which encode the same toxins or which encode toxins having lower orequivalent pesticidal activity. As used here, the term “equivalentpesticidal activity” refers to toxins having similar or essentially thesame biological activity against the target pests as the claimed toxins.

It is well within the skill of a person trained in the art to createalternative DNA sequences encoding the same or essentially the same,toxin. These variant DNA sequences are within the scope of the subjectinvention. As used herein, reference to “essentially the same” sequencerefers to sequences, which have amino acid substitutions, deletions,additions or insertions, which do not materially affect pesticidalactivity. Fragments retaining pesticidal activity are also included inthis definition.

A novel chimeric toxin of the subject invention has been specificallyexemplified herein. It should be readily apparent that the subjectinvention comprises variants or equivalent toxins (and nucleotidesequences encoding equivalent toxins) having the same or similarpesticidal activity of the exemplified toxin. Equivalent toxins willhave amino acid homology with the exemplified toxin. This amino acidhomology will typically be greater than 75%, preferably be greater than90% and most preferably be greater than 95%. The amino acid homologywill be highest in critical regions of the toxin, which account for thebiological activity or are involved in the determination ofthree-dimensional configuration, which ultimately is responsible for thebiological activity. In this regard, certain amino acid substitutionsare acceptable and can be expected if these substitutions are inregions, which are not critical in biological activity or areconservative amino acid substitutions, which do not affect thethree-dimensional configuration of the molecule. For example, aminoacids may be placed in the following classes: non-polar, unchargedpolar, basic and acidic. Conservative substitutions whereby an aminoacid of one class is replaced with another amino acid of the same classfall within the scope of the subject invention so long as thesubstitution does not materially alter the biological activity of theδ-endotoxin. Table 3 as given here below provides a listing of examplesof amino acids belonging to each class.

TABLE 3 Class of Amino Acid Examples of Amino Acids Non-polar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr,Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

In some instances, non-conservative substitutions can also be made. Thecritical factor is that these substitutions must not significantlydetract from the biological activity of the toxin. It is well within theskill of a person trained in the art of protein engineering tosubstitute any amino acid of the chimeric toxin with alanine. Thesubstitution of any amino acid is safest, as alanine is a typical aminoacid. Such substitution is also well within the scope of the invention.

A gene encoding the chimeric toxin of the subject invention can beintroduced into a wide variety of microbial or plant hosts. Expressionof the toxin gene results, directly or indirectly, in the intracellularproduction and maintenance of the pesticidal chimeric toxin. Withsuitable microbial hosts, e.g., Pseudomonas, the microbes can be appliedto the sites where the pests proliferate. This will result into thecontrol of the pest. Alternatively, the microbe hosting the toxin genecan be treated under conditions that prolong the activity of the toxinand stabilize the cell. The treated cell, which retains the toxicactivity, then can be applied to the environment of the target pest.Where the gene encoding the chimeric toxin is introduced via a suitablevector into a microbial host, and said host is applied to theenvironment in a living state, it is essential that certain hostmicrobes be used. Microorganism hosts are selected which are known tooccupy the “phytosphere” (phylloplane, phyllosphere, rhizosphere, and/orrhizoplane) of one or more crops of interest. These microorganisms areselected so as to be capable of successfully competing in the particularenvironment (crop and other insect habitats) with the wild-typemicroorganisms, provide for stable maintenance and expression of thegene expressing the polypeptide pesticide, and, desirably, provide forimproved protection of the pesticide from environmental degradation andinactivation.

A large number of microorganisms are known to inhabit the phylloplane(the surface of the plant leaves) and/or the rhizosphere (the soilsurrounding plant roots) of a wide variety of important crops. Thesemicroorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., genera Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes;fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula and Aureobasidium. Of theparticular interest are such phytosphere bacterial species asPseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonasspheroids, Xanthomonas campestris, Rhozobium melioti, Alcaligenesentrophus and Azotobacter vinlandii and phytosphere yeast species suchas Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei,S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus,Kluyveromyces veronae and Aureobasidium pollulans. of particularinterest are the pigmented microorganisms. A wide variety of ways areavailable for introducing a gene encoding a chimeric toxin into amicroorganism host under conditions, which allow for the stablemaintenance and expression of the gene. These methods are well known tothose skilled in the art and are described, for example, in U.S. Pat.No. 5,135,867, which is incorporated herein by reference.

A plant transformation vector was constructed for the development oftransgenic plants. A plasmid pPK58 (having CaMV35S promoter withduplicated enhancer) was digested with BamHI and HindIII and pPK200 withHindIII and EcoRI to excise out CaMV35S promoter with the duplicatedenhancer and chimeric cry1E gene, respectively. A triple ligation wascarried for cloning of the two fragments in pLITMUS38 cloning vector(New England Biolabs). The plasmid, namely pPK59 had CaMV35S promoterwith the duplicated enhancer at the upstream of the chimeric gene.Restriction analysis of pPK59 is shown in FIG. 2. The nos transcriptionterminator was cloned at the downstream of the chimeric gene. DNA of nospolyadenylation element was amplified using pBI101.1 as template withsuitable primers, which created MeI and EcoRI restriction sites at theupstream and downstream, respectively. The plasmid pPK59 was digestedwith EcoRI and the PCR product was cloned following digestion with theMfeI and EcoRI restriction enzymes. The clone in which EcoRI restrictionsite of the synthetic gene was ligated to MfeI site of nos terminator(as they have compatible ends), selected and named as pPK201. Thecorrect orientation of nos terminator was confirmed by restrictionanalysis and also by sequencing. The expression cassette (the syntheticcry gene with E-35S promoter and nos terminator) was cloned in Ti binaryvector. BamHI-EcoRI fragment of plasmid pPK201 was cloned in pBI101.1replacing BamHI-EcoRI fragment (uidA gene and nos terminator) of theplasmid. This binary vector was named as pPK202. The map of the E. coliand plant expression vectors are shown in FIG. 3.

In order to study the efficacy of the chimeric Cry1E toxin in plants,tobacco was selected for the expression. Agrobacterium tumefaciensstrain LBA 4404 containing the helper plasmid pAL4404 was transformedwith the binary vector pPK202 following the modified protocol of“electroporation of Agrobacterium” discussed by Cangelosi et al. (1991)and transformed colony was selected on antibiotics streptomycin,rifampicin and kanamycin. Agrobacterium mediated transformation ofNicotiana tobacum cv. Patit Havana was carried out following the methodof Horsch et al., 1985 and the transgenic plant were selected on theantibiotic kanamycin. The presence of the gene encoding chimeric toxinwas confirmed with PCR and Southern analysis and the expression of theto transgene was established with the RT-PCR, Western analysis andELISA. ELISA result displayed 0.5% expression of the toxin protein outof total soluble leaf protein in a selected transgenic line. This highlevel of the expression was the result of the designing of the gene inwhich plant-preferred codons were exclusively used. Plant preferredtranslation initiation context used in this study would also have playedan important role in achieving enhanced expression.

Insect bioassay was performed with two months old transgenic plants and1^(st) and 5^(th) instar larvae of Spodoptera litura. (Please referFIGS. 4-6). 15 cm² leaf-discs of transgenic and control plants wereplaced in cylindrical boxes containing wet blotting paper at the bottomand ten 1^(st) instar larvae were released onto them. Mouths of theboxes were covered with wet muslin cloth to maintain sufficient humidityand exchange of air. The toxicity experiments were conducted in threereplicates at 25±0.2° C. and 16/8 h photoperiod was maintained. Inbioassay with 5^(th) instar larvae, complete leaves of transgenic andcontrol plants were used. The leaf petiole was held in cotton plug overa 250 ml flask containing ½ MS salt solution to overcome wilting of theleaf. Five insect larvae were allowed to feed on each leaf. The leavesof control plant were changed after every 8 h and they were consumedcompletely by the insect larvae.

The result (FIGS. 4 and 6) established that 1^(st) as well as 5^(th)instar larvae died within 48-72 h. The amount of leaf eaten by the1^(st) instar larvae was negligible as compare to the control plant leafdiscs, which were eaten voraciously. Ingestion of approximately 1 cm² oftransgenic leaf was sufficient to kill 5^(th) instar larvae. Theselarvae appeared moribund after 8-16 h of feeding on the transgenic leafand finally died within 2-3 days. Green coloured excreta with high watercontent were noticed on leaf surface. Some of the larvae showed heavyweight loss before death. In a separate experiment, different instarlarvae were allowed to feed on leaves of transgenic plant for 1 h, 2 h,4 h, 8 h and 16 h and then shifted to control plant leaves. It wasobserved that 8 h of feeding on transgenic plants was sufficient tocause 100% mortality of larvae in all stages of development, even afterfeeding on non-transgenic plants. Ingestion of very small amounts of thetoxin by young larvae delayed their pupation by 10-15 days from normallarval cycle of 15 days. The few larvae that escaped mortality developedinto flies. 40% of the paired matings where such flies were used, gaveeggs. However, the eggs were sterile and failed to hatch. The totalsoluble protein from transgenic tobacco plant was extracted and loadedon SepharoseQ ion exchange column. The protein was eluted withincreasing gradient of sodium chloride and the peak containingδ-endotoxin was pooled and desalted on G10 column. The eluted proteinwas mixed in semi-synthetic diet. Similar protein extraction andpurification from the leaf of non-transgenic tobacco plants was alsoperformed and such plant protein served as control. The toxicityexperiment was conducted as discussed earlier. EC₅₀ for Spodopteralitura and Helicoverpa armigera was 37 ng/ml and 285 ng/ml of artificialdiet. The result again confirmed the efficacy of chimeric δ-endotoxintowards target insect pests.

A novel δ-endotoxin for the control insect pests and a gene for its highlevel expression in plants, which comprises theoretical designing of anovel δ-endotoxin, herein named chimeric Cry1E, strategically designedby replacing a polypeptide domain (from position 530 to 587) of Cry1Eaprotein by that of Cry1Ca (from position 533 to 602), a novel 25 aminoacid residues long polypeptide at the C-terminus extreme of the proteinfor stability, theoretical designing of the gene to express the chimericδ-endotoxin at a high level in plants, designing and chemical synthesisof the oligonucleotides representing the theoretically designed gene,assembly of oligonucleotides into double stranded DNA, cloning andsequence analyses of the cloned synthetic DNA, construction of vectorsfor the expression of chimeric gene in E. coli and plants, expression ofthe synthetic gene in E. coli, comparison of the toxicity of thechimeric protein with the parental proteins against Spodoptera litura,transformation of plant, for example tobacco with the chimeric gene,high level expression of the engineered protein in transgenic plants andevaluation of the potential of the chimeric toxin in transgenic plantsfor protection against Spodoptera litura.

In still another embodiment of the present invention, wherein amino acidno. 1 to 529 of Cry1Ea, 530 to 599 of Cry1Ca, 600 to 616 of Cry1Ea and617 to 641 a novel polypeptide and its structurally and/or functionallyequivalent variants or fragments thereof.

In still another embodiment of the present invention, wherein the saidtoxin has an amino, acid sequence shown in SEQ ID No.1; shows severalfold higher toxicity to target lepidopterin insects as compared to theparental toxins Cry1Ea and Cry1Ca.

In still another embodiment of the present invention, wherein chimericcry1E gene which encodes a chimeric toxin having activity againstlepidopteran insects, has nucleotide sequence shown in SEQ ID No 2 orits fragment thereof.

In still another embodiment of the present invention, wherein a processof controlling lepidopteran pests employing the protein, with aneffective amount of the toxin used as such or as a component of achemical or microbial formulation.

In still another embodiment of the present invention, wherein arecombinant DNA transfer vector comprising a polynucleotide sequence,which encodes a toxin having activity against lepidopteran insects,wherein said polynucleotide sequence has the nucleotide sequence of SEQID NO. 2 or fragments thereof.

In still another embodiment of the present invention, wherein arecombinant host transformed with the gene.

In still another embodiment of the present invention, wherein the saidhost is a microbe for example Escherichia coli, Pseudomonas, Yeast,Cyanobacteria and/or other microbes.

In still another embodiment of the present invention, wherein saidtransformed host is a plant for example tobacco, cotton, chickpea,pegeonpea, groundnut, cauliflower, cabbage, chilli, capsicum and/orother plants, which expresses the toxin, wherein said toxin has theamino acid sequence of SEQ ID NO. 1, or its variants with equivalentactivity against lepidopteran insects.

In further embodiment of the present invention, instant Applicationclearly states that in proteins, more particularly in the field ofendotoxins, the high homology of the sequence is not found to make anysignificant difference in activity. The above-referred work on ofendotoxins Cry1Aa1 to Cry1Aa6 clearly reflect the essence of this work.In the instant Application, the applicant has observed extraordinarilyhigh insecticidal activity. Further, the homology of 70% and above inthe sequence of chimeric protein Cry1E of the instant Application isalso found to show no significant change in the activity. This meansthat the proteins with sequence homology of 70% and above for chimericprotein Cry 1E are used as insecticidal agents.

The following examples are given by way of illustrations and thereforeshould not be construed to limit the scope of the present invention.

Example 1 Comparative Toxicity of the Novel Chimeric Cry1E Expressed inE. coli to Larvae of Lepidopteran Insect Pests

A chimeric δ-endotoxin, 616 amino acid residues long, herein saidchimeric Cry1E was strategically designed by replacing a polypeptidedomain (from position 530 to 587) of Cry1Ea protein by that of Cry1Ca(from position 533 to 602). A polypeptide of 25 amino acid residues wasadditionally included at the C-terminus extreme as described earlier. A1.99 kb nucleotide sequence was theoretically designed to code for theabove-mentioned chimeric δ-endotoxin. The codons for each amino acidwere distributed evenly to avoid temporary deficiency of the tRNA duringtranslation. Several 6-base cutter restriction enzyme sites were createdin the designed gene. BamHI, HindIII and NcoI restriction sites werecreated at 5′-end and EcoRI at the 3′-end of the designed gene. The genewas divided into 58 overlapping oligonucleotides (40 to 65 nucleotideslong). Each oligonucleotide had 13-18 nucleotide long overlap with theimmediately adjacent oligonucleotides on the complementary strand (T_(m)between 48-50° C.). Oligonucleotides were synthesised on a DNAsynthesiser (Gene Assembler Special, Pharmacia, Sweden) at 200 nmolescale and purified on denaturing urea-PAGE. All 58 oligonucleotides wereassembled into the double-stranded DNA, herein said chimeric cry1E genefollowing the ligation-free gene synthesis method of Singh et al. (1996)and as shown in FIG. 1. The DNA was digested with HindIII and EcoRIrestriction enzyme and cloned in pBluescriptII SK(+) (Stratagene). Theplasmid was named as pPK200. The nucleotide sequence of the syntheticDNA was confirmed by sequencing of the cloned synthetic DNA on automatedDNA sequencing system (Applied Biosystems model 373).

A cassette was constructed for the expression of the chimeric toxin inE. coli under control of T7 promoter. For this, plasmid pPK200 wasdigested with the restriction enzymes NcoI and BanHI and cloned inexpression vector pET-19b (Novagen). The plasmid was named as pPK206.DNA encoding toxin portion of Cry1Ea and Cry1Ca were amplified withpolymerase chain reaction, using suitable primers, which created NcoIand BamHI restriction sites at the upstream and the downstream of theamplicon, respectively in both the DNA. The amplified products werecloned at NcoI and BamHI site in the same vector (pET-19b). Theconstructs having Cry1Ea toxin DNA was named as pPK141 and Cry1Ca aspPK135 as described earlier. BL21DE3 strain of E. coli was transformedwith the constructs pPK141, pPK135 and pPK206. The toxin proteins wereexpressed by induction with appropriate concentrations of IPTG. Theexpression was carried out at 15° C. to avoid any possible mis-foldingof the toxins. The toxin proteins were quantified densitometrically onthe denaturing polyacrylamide gel. Serial dilutions of the toxins weremixed in semi-synthetic diet. Total E. coli protein served as control inthe diet. Fifteen neonatal larvae of Spodoptera litura were releasedonto the cakes of the diet mixture in a 100-ml beaker and the mouth ofthe beaker was covered with muslin cloth to allow gas exchange. Eachexperiment was conducted in 6 replicates. The diet was changed afterevery alternate day. Bio-assay was conducted with 16/8 h photoperiod at25±0.2° C. Toxicity data was recorded after 7 days of the feeding. EC₅₀was determined by standard log-probit analysis. All three proteins weretested simultaneously. The representative results are presented in Table4 as given here below.

TABLE 4 δ-endotoxins(S) EC₅₀ (μg/ml semi-synthetic diet) Cry1Ea >108Cry1Ca 29.48 ± 1.77 Chimeric Cry1E  6.27 ± 0.59

The result showed that the chimeric toxin was several fold more toxicover Cry1Ea and more than four fold toxic over Cry1Ca protein. Cry1Eatoxin protein failed to cause any mortality or growth retardation of theSpodoptera larvae. The result established the successful engineering ofthe Cry1Ea toxin for converting it into a biologically active improvedtoxin. The engineered protein was more toxic than Cry1Ca protein, whichis the best-known δ-endotoxoin against Spodoptera sp.

A similar toxicity experiment was conducted with the larvae ofHelicoverpa armigera. In this case, 72 h old larvae were released onsemi-synthetic diet containing one of the three proteins and only onelarvae was released in each box. Weight loss was recorded after 7 daysof feeding. The representative results are presented in Table 5 as shownhere below.

TABLE 5 δ-endotoxins(S) EC₅₀ (μg/ml semisynthetic diet) Cry1Ea >176Cry1Ca 136.22 ± 8.77 Chimeric Cry1E  26.71 ± 1.39

The result shows that the novel chimeric 8-endotoxoin designed by us isnot only more toxic to Spodoptera but also effective againstHelicoverpa. The designing has widened the host range of the toxin aswell as substantially improved toxicity over the parental proteins.

Example 2 High Larval Toxicity of the Transgenic Plants Expressing NovelChimeric Cry1E Protein

In order to establish efficacy of the novel chimeric toxin in plants, a,plant transformation vector was constructed for the development oftransgenic plants. A plasmid pPK58 (having CaMV35S promoter withduplicated enhancer) was digested with BamHI and HindIII and pPK200 withHindIII and EcoRI to excise out CaMV35S promoter with the duplicatedenhancer and chimeric cry1E gene, respectively. A triple ligation wascarried for the cloning of the two fragments in pLITMUS38 cloning vector(New England Biolabs). The plasmid was named pPK59, which had CaMV35Spromoter with the duplicated enhancer at the upstream of the chimericgene. The nos transcription terminator was cloned at the downstream ofthe chimeric gene nos polyadenylation element was amplified usingpBI101.1 as template with suitable primers, which created MfeI and EcoRIrestriction sites at the upstream and downstream, respectively. Theplasmid pPK59 was digested with EcoRI and the PCR product was clonedfollowing the digestion with the MfeI and EcoRI restriction enzymes. Theclone in which EcoRI restriction site of the synthetic gene ligated toMfeI site of nos terminator (as they have compatible ends) was selectedand named as pPK201. The correct orientation of nos terminator wasconfirmed by restriction analysis and also by DNA sequencing. Theexpression cassette (the synthetic cry gene with E-35S promoter and nosterminator) was cloned in Ti binary vector. BamHI-EcoRI fragment ofplasmid pPK201 was cloned in pBI101.1 replacing BamHI-EcoRI fragment(uidA gene and nos terminator) of the plasmid. This binary vector wasnamed as pPK202. The construction of E. coli and plant expression vectoris schematically presented in FIG. 3. The construct had polynucleotidesequences TAAACCATG GCT (SEQ ID NO:9) as plant preferred translationinitiation context, TAA TGA were introduced in synthetic gene fortranslational termination. Agrobacterium tumefaciens strain LISA 4404containing helper plasmid pAL4404 was transformed with binary vectorpPK202 following the modified protocol of “electroporation ofAgrobacterium” discussed by Cangelosi et al. (1991) and transformedcolony was selected on antibiotics streptomycin, rifampicin andkanamycin. Agrobacterium mediated transformation of Nicotiana tabacumcv. Patit Havana was carried out following the method of Horsch et al.,1985 and the transgenic plant were selected on the antibiotic kanamycin.The presence of the gene encoding chimeric toxin was confirmed with thePCR and Southern Analysis and the expression of the transgene wasestablished with the RT-PCR, Western analysis and ELISA. ELISA resultestablished 0.5% expression of the toxin protein in total soluble leafprotein in the transgenic line selected for these experiments. This highlevel of the expression was the result of the designing of the gene inwhich plant-preferred codons were exclusively used. Plant preferredtranslation initiation context also would have played an important rolein the expression. Insect bioassay was performed with the leaves oftwo-month-old transgenic plants and neonatal larvae of Spodopteralitura. 15 cm² leaf-discs of transgenic and control plants were placedin cylindrical boxes containing wet blotting paper at the bottom and ten1^(st) instar larvae were released onto them. Mouths of the boxes werecovered with wet muslin cloth to maintain sufficient humidity and toallow the exchange of air. The toxicity experiments were conducted insix replicates at 25±0.2° C. and 16/8 h photoperiod was maintained. Theresult showed (FIG. 4) that the transgenic plants expressing novelchimeric protein were highly toxic to the neonatal larvae of Spodopteralitura and cause 100% mortality within 48 h of feeding. The damage ofleaf-discs by the insect larvae was negligible as compared to controlplant leaf-discs, which were almost completely eaten away. High level ofthe protection of the transgenic plant and mortality of Spodopteralarvae upon feeding on transgenic plants again established the efficacyof the chimeric toxin and the transgenic plants.

Example 3 High Toxicity of the Chimeric Cry1E Protein to Larvae ofSpodoptera sp. in all the Stages of their Development

A bioassay was conducted on 1^(st) (3 days old), 3^(rd) (7 days old) and5^(th) (12 days old) instar larvae to established the efficacy of theengineered protein expressed in the transgenic plants. Bioassay with1^(st) instar larvae has been discussed in example 2. Complete leaves oftransgenic and control plants were used for feeding the advanced stagelarvae. The leaf petiole was held in cotton plug over a 250 ml flaskcontaining ½ MS salt solution to overcome wilting of the leaf. 5 insectlarvae were allowed to feed on each leaf. The leaves of control plantfed by 3^(rd) (FIG. 5) and 5^(th) (FIG. 6) instar larvae were changedafter 16 h and 8 h, respectively, as they were consumed completely bythe insect larvae. The result established that feeding on transgenicleaf causes mortality of larvae in all the developmental stages within48 h. Ingestion of approximately 1 cm' of transgenic leaf was sufficientto kill 5^(th) instar larvae. These larvae appeared moribund after 8-16h of feeding on the transgenic leaf and finally died within 2 days.Green coloured excreta with high water content were noticed on leafsurface. Some of the larvae showed heavy weight loss before death.

In a separate experiment, different instar larvae were fed on leaves oftransgenic plants for 1 h, 2 h, 4 h, 8 h and 16 h and then shifted tocontrol plant leaves. It was observed that 4 h of feeding on transgenicplants was sufficient to cause 100% mortality of larvae in all stages ofdevelopment, even when they were subsequently fed on non-transgenicplants. Ingestion of very small amounts of the toxin by young larvaedelayed their pupation by 10-15 days beyond the normal larval cycle of15 days. The few larvae that escaped mortality developed into flies. 40%of the paired matings using such flies gave eggs. However, the eggs weresterile and failed to hatch. The toxicity of δ-endotoxin to advancestage insect larvae has not been reported in literature till date. Thehigh level of toxicity may be due to higher stability of chimeric toxinin the mid gut of insect larvae or improved receptor binding andpore-forming ability of δ-endotoxin. The example again established thepotential of the chimeric toxin against Spodoptera sp. Since Helicoverpadoes not prefer to cat tobacco leaf, toxicity experiment with transgenictobacco plants could not be conducted on Helicoverpa.

Example 4 High Toxicity of the Novel Chimeric Cry1E Prepared FrontLeaves of Transgenic Tobacco Plants Expressing δ-Endotoxin

Total soluble protein was prepared from leaves of transgenic tobacco.Fresh leaf tissue was powered under liquid N2 and then suspended in 5volumes of protein extraction buffer (TrisCl, 20 mM, pH 9.5; EDTA 2 mM,pH 8.0; NaCl, 50 mM; DTT, 1 mM; PVP 2% and PMSF, 100 mM). The suspensionwas mixed well and centrifuged twice (20,000×g, 20 min and 4° C.). Thesupernatant was loaded on Sepharose Q column (10 cm×2.5 cm). The proteinwas eluted with increasing gradient of NaCl in extraction buffer and 100fractions of 5 ml were collected. The δ-endotoxin was detected withELISA. The fractions containing the δ-endotoxin were pooled. A knownamount of the plant-purified δ-endotoxin was mixed in semi-syntheticdiet. The toxicity trials were conducted with 3-day old larvae ofSpodoptera litura and Helicoverpa armigera, as described in previousexamples. The result showed that EC₅₀ (the concentration required for50% killing) for Spodoptera and Helicoverpa were 42.39±1.72 ng/ml and283.11±8.29 ng/ml of semi-synthetic diet. The result further establishedthe high level of toxicity to the larvae of Spodoptera and Helicoverpa.The point is noteworthy that insecticidal crystal protein made in planttissue is much more toxic as compared to the same protein made in E.coli. Probably the δ-endotoxin folds much better in plant cytoplasm, therole of some unidentified chaperons in such folding cannot be overruled.

Example 5 Stability of Chimeric δ-Endotoxin in Plant Tissue

Total soluble leaf protein of transgenic plant was extracted asdiscussed earlier and incubated at 4° C. and 28° C. They were used fortoxicity trials. The samples were taken out after every two days in caseof former and every day in case of later. The total crude protein wasmixed in semi synthetic diet and toxicity experiment was carried outwith neonatal larvae of Spodoptera litura. The insect mortality data wasrecorded after 7 days of feeding and LC₅₀ was calculated. The resultsare shown in Tables 6, and 7 here below.

TABLE 6 Incubation period of crude protein LD₅₀ (in ng/ml of semi- S.No. at 4° C. (in days) synthetic diet) 1. 2 48.71 ± 2.4  2. 4 57.87 ±2.96 3. 6 66.44 ± 3.65 4. 8 70.19 ± 3.55 5. 12 73.82 ± 3.1  6. 16 74.37± 3.67

TABLE 7 Incubation period of crude protein LD₅₀ (in ng/ml of semi- S.No. at 28° C. (in days) synthetic diet) 1. 1 76.44 ± 3.3 2. 2 98.13 ±4.6 3. 3 143.46 ± 8.5  4. 4 — 5. 5 —

The result established the stability of chimeric δ-endotoxin designed byus against the plant proteases. The chimeric toxin was stable for morethan 16 days at 4° C. and 3 days at 28° C., and caused more than 80%mortality. Increase in LC₅₀ with time was presumably due to somedegradation of the toxin.

Main advantages of the present invention are:

-   1. Cry1Ea δ-endotoxin was engineered to obtain a novel chimeric    toxin, herein said chimeric Cry1E. The toxicity of the chimeric    protein was several fold higher as compared to the parent toxins or    other δ-endotoxin reported to function against Spodoptera. Its    larvicidal activity was very high when it is made in plants.-   2. The gene encoding chimeric toxin was designed to express both    in E. coli and plants. Hence, it can be used in the engineering of a    microbe for the expression of chimeric toxin, which can be used in    the preparation of the microbial formulation. The same gene can also    be used in the genetic engineering of the plants for the trade of    insect resistance. We have shown that the sequence designed by us    gives very high level of expression (0.5% of the total soluble    protein) of the chimeric toxin in transgenic tobacco and cotton    leaves (results not included).-   3. The transgenic plants expressing the chimeric toxin exhibited    very high degree of protection against the larvae of Spodoptera    litura in all developmental stages. They died within 2-3 days of    feeding on the transgenic plants. Such high level of toxicity of    transgenic plants against any lepidopteran insect has not been    reported till date. This protein may also be effective against many    other insect larvae. Our results show that the toxin was effective    against Helicoverpa also.-   4. Potential of the chimeric toxin in transgenic plants was further    established by short-term feeding on transgenic plants. Feeding for    4 hours caused 100% mortality of Spodoptera larvae at all the    developmental stages. The feeding for extremely short (up to one    hour) periods, delayed larval development and interfered with    metamorphosis. Such protein may be extremely valuable in protecting    agronomically important crops and forests. The gene coding chimeric    toxin can be used in the development of transgenic plants and/or for    production of the toxin in a microbe, which can be used in microbial    formulations.-   5. Since the transgenic plants expressing the novel chimeric toxin    caused 100% mortality of Spodoptera larvae within a very short    period of feeding, the probability of the development of resistance    in insects against this δ-endotoxin will be extremely low.

1-41. (canceled)
 42. A chimeric δ-endotoxin protein comprising an aminoacid sequence that is identical to amino acids 530-641 of SEQ ID NO:1,said chimeric protein having greater toxicity than Cry1C as determinedby EC₅₀ in a challenge to neonatal Spodoptera litura larvae.
 43. Achimeric δ-endotoxin protein comprising an amino acid sequence that isat least 95% identical to amino acids 1-641 of SEQ ID NO:1, said proteinhaving a sequence identical to amino acids 530-641 of SEQ ID NO:1, saidchimeric protein having greater toxicity than Cry1C as determined byEC₅₀ in a challenge to neonatal Spodoptera litura larvae.
 44. Theprotein as claimed in claim 42 or 43, wherein said chimeric protein isstable at temperature ranging between 4-35° C.